Divergent strategies of photoprotection in high-mountain plants

Transcription

1 Planta (1998) 207: 313±324 Divergent strategies of photoprotection in high-mountain plants P. Streb 1, W. Shang 1, J. Feierabend 1, R. Bligny 2 1 Botanisches Institut, J.W. Goethe-UniversitaÈ t, Postfach , D Frankfurt am Main, Germany 2 Laboratoire de Physiologie Cellulaire Ve geâ tale, DBMS, Unite de Recherche Associe e au Centre National de la Recherche Scienti que 576 et Universite Joseph Fourier, Commissariat aá l'energie Atomique, 17 rue des Martyrs, F Grenoble Cedex 9, France Received: 18 March 1998 / Accepted: 7 August 1998 Abbreviations and symbols: DTT ˆ dithiothreitol; F v /F m ˆ ratio of variable to maximum chlorophyll a uorescence; PAR ˆ photosynthetically active radiation; PPT ˆ phosphinothricin; Q A ˆ primary electron acceptor of PSII; qn ˆ coe cient of non-photochemical quenching of uorescence yield; qp ˆ coe cient of photochemical quenching of uorescence yield Correspondence to: J. Feierabend; Feierabend@em.uni-frankfurt.de; Fax: 49 (69) Abstract. Leaves of high-mountain plants were highly resistant to photoinhibitory damage at low temperature. The roles of di erent photoprotective mechanisms were compared. Mainly, the alpine species Ranunculus glacialis (L.) and Soldanella alpina were investigated because they appeared to apply greatly divergent strategies of adaptation. The ratio of electron transport rates of photosystem II/photosystem I measured in thylakoids from R. glacialis did not indicate a speci c acclimation to high irradiance. Low rates of a chloroplast-mediated inactivation of catalase (EC ) in red light indicated, however, that less reactive oxygen was released by isolated chloroplasts from R. glacialis than by chloroplasts from lowland plants. Leaves of S. alpina and of Homogyne alpina (L.) Cass, but not those of R. glacialis, had a very high capacity for antioxidative protection, relative to lowland plants, as indicated by a much higher tolerance against paraquatmediated photooxidative damage and a higher a-tocopherol content. Accordingly, ascorbate and glutathione were strongly oxidized and already largely destroyed at low paraquat concentrations in leaves of R. glacialis, but were much less a ected in leaves of S. alpina. Nonradiative dissipation of excitation energy was essential for photoprotection of leaves of S. alpina and depended on the operation of the xanthophyll cycle. Strong nonphotochemical quenching of chlorophyll uorescence occurred also in R. glacialis leaves at high irradiance, but was largely independent of the presence of zeaxanthin or antheraxanthin. For R. glacialis, photorespiration appeared to provide a strong electron sink and a most essential means of photoprotection, even at low temperature. Application of phosphinothricin, which interferes with photorespiration by inhibition of glutamine synthetase, caused a striking reduction of electron transport through photosystem II and induced marked photoinhibition at both ambient and low temperature in leaves of R. glacialis, while S. alpina was less a ected. Key words: Alpine plant ± Antioxidant ± Paraquat tolerance ± Photoinhibition ± Photorespiration ± Xanthophyll cycle Introduction High-mountain plants depend on a highly e cient carbon assimilation since their growing season is very short. Although the extreme climatic conditions with extended periods of high irradiance and low temperature, rapid temperature changes, and a reduced partial pressure of CO 2 are not favorable for photosynthesis, the photosynthetic performance of alpine plants appears to be well adapted to the environment. Previous studies have shown that in several alpine plants photosynthesis is highly e cient at cool temperatures and also adapted to high irradiance (Moser et al. 1977; KoÈ rner and Larcher 1988). Highly sensitive and widespread symptoms of photodamage in non-adapted plants are the photoinactivation of photosystem II (PSII) and of the peroxisomal enzyme catalase. The reaction-centre protein D1 of PSII and catalase usually have a rapid turnover in light and therefore require continuous repair by new synthesis. (Hertwig et al. 1992; Aro et al. 1993, Foyer et al. 1994; Feierabend et al. 1996). In nonadapted plants the apparent loss of both PSII and catalase activity by photoinactivation is greatly enhanced at low temperature where protein synthesis is suppressed. (Feierabend et al. 1992; Huner et al. 1993; Wise 1995). However, in leaves of the alpine plants, Homogyne alpina, Soldanella alpina and Ranunculus glacialis, PSII and catalase are highly resistant to

2 314 P. Streb et al: Photoprotection in alpine plants photoinhibitory damage even when protein synthesis is suppressed by inhibitors of translation or by exposure to low temperature (Streb et al. 1997). The results of previous investigations (Streb et al. 1997) suggested that the maintenance of stable levels of the D1 protein of PSII (Shang and Feierabend 1998) and of catalase did not greatly depend on repair by new protein synthesis. However, in most instances this high stability was only observed in intact leaves. In vitro, both PSII of isolated thylakoids from H. alpina and R. glacialis and catalase in cell-free leaf extracts from S. alpina and R. glacialis were as rapidly photoinactivated as known for lowelevation plants. The only exception was a catalase from H. alpina which after extraction also had a much higher stability in light than other catalases (Streb et al. 1997). In all other instances the high resistance to photoinactivation of PSII and catalase must depend on the presence of more e cient mechanisms of protection against oxidative damage in leaves of the alpine plants than in non-adapted low-elevation plants. Unexpectedly, the strategies of protection appear to be very divergent in the alpine plants H. alpina, R. glacialis and S. alpina (Streb et al. 1997). Important mechanisms for the avoidance of reactive-oxygen production in chloroplasts, or for its removal, are the thermal dissipation of excitation energy which has frequently been correlated with the light-dependent formation of zeaxanthin in the xanthophyll cycle (Demmig-Adams and Adams III 1996), superoxide dismutase, and antioxidants and antioxidative enzymes of the ascorbate-glutathione cycle (Asada 1994; Foyer et al. 1994). Accordingly, high contents of xanthophyllcycle pigments, of the antioxidants ascorbate and glutathione, and of antioxidative enzymes have usually been observed in cold-adapted plants (SchoÈ ner and Krause 1990; Wise 1995; Thiele et al. 1996; Leipner et al. 1997), and the levels of these compounds and enzymes are also extraordinarily high in leaves of the alpine plant S. alpina but not in those of H. alpina and R. glacialis (Wildi and LuÈ tz 1996; Streb et al. 1997). While the occurrence of a light-stable catalase might contribute to the light-stress tolerance of H. alpina, the very poor antioxidative systems in R. glacialis are unusual. Therefore, we have now further evaluated the relevance of various protective mechanisms to the lightstress tolerance of alpine plants and mainly concentrated on a comparison of the two most divergent species S. alpina and R. glacialis. Where appropriate, properties of the alpine plants were compared with those of lowland plants that were either collected from eld sites (Ranunculus, Taraxacum) or grow under controlled laboratory conditions. Chloroplasts were analysed for potential functional adaptations. In those plants that allowed the isolation of intact organelles in su cient yields. The overall strength of antioxidative protection was examined by a comparison of the sensitivity of the leaves to paraquat which mediates superoxide formation by electron transfer from PSI to O 2 and thus greatly enhances photooxidative damage (Dodge 1994). The role of the xanthophyll-cycle pigments was analysed by application of dithiothreitol (DTT) which inhibits the formation of zeaxanthin from violaxanthin (Demmig-Adams et al. 1990). Further, the herbicide phosphinothricin (PPT) was applied as a tool to assess the role of photorespiration. Phosphinothricin irreversibly inhibits glutamine synthetase and prevents the reassimilation of ammonia. As a result, photorespiration is suppressed because glutamate is lacking for the transamination of glyoxylate to glycine (Wendler and Wild 1990; Wendler et al. 1990; Kozaki and Takeba 1996). Photorespiration appears to provide an essential mechanism to protect the photosynthetic apparatus against photooxidative damage under strong irradiance (Wu et al. 1991; Heber et al. 1996; Kozaki and Takeba 1996). Materials and methods Plant materials and eld sites. Alpine plants were investigated during the months June and July in the years 1996 and 1997 in the laboratories of the Station Alpine du Lautaret of the University Joseph Fourier of Grenoble at the Col du Lautaret (2100 m altitude) in the western part of the French Alps. Leaves of Soldanella alpina L., Homogyne alpina (L.) Cass and Ranunculus glacialis L. were collected from the same eld sites at 2400±2700 m between the Lautaret and the Galibier pass that were described by Streb et al. (1997). Mean daily temperatures and light intensities during the investigation period were as previously reported (Dorne et al. 1986; Streb et al 1997). For comparison, fully expanded mature leaves of the non-alpine plants Taraxacum o cinale Wiggers and Ranunculus acris L. were collected from unshaded sites of the Botanical Garden of the University of Frankfurt am Main during the months July till October. Some experiments were performed with primary leaves of rye plants (Secale cereale L. cv. Halo) grown under constant conditions either for 6 d at 22 C or for 5 weeks at 4 C and a photon ux of 96 lmol m )2 s )1 photosynthetically active radiation (PAR) in the presence of a modi ed nutrient solution, as previously described (Streb et al. 1993). Experimental treatments and light exposure. For experimental treatments under controlled conditions, incident light of 500± 2000 lmol m )2 s )1 PAR was generated with halogen lamps (20 W, 75 W and 100 W). The intensity of sunlight was not constant during the incubation periods. Incident light varied between 650 and 2350 lmol m )2 s )1 PAR, with a mean photon ux of 1800 lmol m )2 s )1 PAR. Periods of incident light of lower than 1500 lmol m )2 s )1 PAR were short, resulting from a few clouds during the course of the experiments. The ambient temperature during the incubations in sunlight ranged from 10 C to 27 C with a mean temperature of 18 C. When incubations were performed in an ice bath, the temperature observed in sunlight at the leaf surface ranged between 4 C and 9 C, with a mean temperature of 7 C. For incubations in sunlight, di erential treatments were always performed in parallel, in order to exclude e ects caused by variations of light or temperature conditions that might occur on di erent days of exposure. For incubations in the presence of paraquat, leaves were collected in the morning. Leaves were oated in Petri dishes on water or paraquat solution and irradiated for 24 h under temperature-controlled conditions at 25 C with white light of 1000 lmol m )2 s )1 PAR. For experiments with DTT, leaves were collected in the evening and incubated overnight for approximately 14 h at 20 C in darkness in Petri dishes on water (controls) or solutions of 3 mm or 30 mm DTT. For experiments with PPT, leaves were collected in the morning and incubated for 2 h in Petri dishes on water or solutions of 1 mm PPT at an ambient temperature of approximately 20 C in weak room light. Subsequent to the latter

3 P. Streb et al: Photoprotection in alpine plants 315 preincubation periods, leaves treated with DTT or PPT and respective controls were exposed to constant light conditions between 500 and 2000 lmol m )2 s )1 PAR at either 25 C or on ice, or to natural sunlight at either ambient temperature or on ice. Isolation of chloroplasts. For the isolation of chloroplasts, leaves were cut to small pieces and nely minced with razor blades under ice-cold conditions, using the grinding medium of ph 6.1 described by Jensen and Bassham (1966), except that KNO 3 was omitted and 10 mm Na 2 S 2 O 4 was used as reducing agent. Alternatively, homogenates without Na 2 S 2 O 4 were prepared in a glove bag under an N 2 atmosphere. The homogenate was passed through four layers of muslin and one layer of Miracloth (Calbiochem) and loaded onto stepped Percoll gradients according to HoÈ inghaus and Feierabend (1983). The gradients consisted of layers of 15%, 42%, and 80% (v/v) Percoll contained in a bu er medium of 0.3 M sucrose, 50 mm Mes-KOH (ph 6.1), 5 mm Na 2 EDTA, 5 mm MnCl 2, 5 mm MgCl 2, and were centrifuged for 5 min at g and 4 C. Intact chloroplasts were collected from the interface of 42% and 80% Percoll. The chloroplast fractions were diluted 2- to 3-fold either with the assay medium for chloroplast-mediated catalase inactivation, consisting of 0.33 M sorbitol, 50 mm Hepes- KOH (ph 7.6), 2 mm Na 2 EDTA, 1 mm MgCl 2, 10 mm KCl or with assay medium used for the estimation of photosynthetic electron transport activities. Chloroplasts were re-pelleted by 5 min centrifugation at 4000 g and 4 C, re-suspended with the respective assay medium and adjusted to a chlorophyll concentration of 1 mg ml )1. Intactness of chloroplasts was examined according to Heber and Santarius (1970) and amounted, on average, to 83%. Chloroplast-mediated catalase inactivation. For the assay of chloroplast-mediated in vitro inactivation of catalase, puri ed bovine liver catalase (10 lkat ml )1, Sigma) was incubated together with a suspension of Percoll-gradient-puri ed intact chloroplasts (50 lg chlorophyll ml )1 ) in the assay medium used for the re-suspension of the chloroplasts. The mixture was incubated in a water bath at 25 C in red light (800 lmol photons m )2 s )1 PAR) provided by Osram halogen lamps in combination with the Schott lters RG 1 (>50% transmission above 610 nm) and KG 3. Analytical methods. Electron transport rates were measured at 25 C with a Hansatech (Kings Lynn, Norfolk, UK) oxygen electrode at saturating light intensity. For the assays the concentrated chloroplast fractions were diluted with H 2 O to a nal chlorophyll concentration of 30 lg ml )1. The PSII electron transport was assayed with phenyl-p-benzoquinone as acceptor according to Hundal et al. (1995). The PSI electron transport from ascorbate to methyl viologen and whole-chain electron transport from H 2 O to methyl viologen were assayed according to Allen and Holmes (1986). All assays contained 5 mm NH 4 Cl. Leaves were extracted by the preparation of homogenates of 5.0 ml nal volume from 0.4 g leaf tissue (fresh weight). Homogenates for catalase (EC ) measurements were prepared in 50 mm K-phosphate bu er (ph 7.5) at 4 C, and enzyme activity was estimated according to Streb et al. (1993). Homogenates for glutamine synthetase (EC ) measurements were prepared according to Nesselhut and Harnischfeger (1981) in 50 mm imidazole bu er (ph 7.5) containing 15 mm MgSO 4, 8 mm 2-mercaptoethanol and 10 mm ATP. The extracts were centrifuged for 30 min at 4 C and g and supernatants were used for the enzyme assays. Glutamine synthetase activity was assayed as the hydroxylamine-dependent synthetase reaction according to Nesselhut and Harnischfeger (1981). An extinction coe cient of e ˆ mm )1 cm )1 was determined at 535 nm for the Fe-glutamylhydroxamate complex. Antioxidants were extracted in 1% (w/v) metaphosphoric acid and analysed by HPLC as described previously (Streb and Feierabend 1996), except that an Eurosphere-100-C18 reversedphase column (Knauer, Berlin, Germany) was used for the separation of ascorbic acid and glutathione. The total chlorophyll content was estimated according to Arnon (1949) in 80% acetone extracts. For analysis by HPLC of pigments and a-tocopherol, approximately 0.4 g of fresh leaf tissue was frozen in liquid nitrogen and stored at )20 C until use. The leaf material was extracted, essentially as described by Thayer and BjoÈ rkman (1990). Frozen leaves were ground with mortar and pestle in liquid nitrogen in the presence of some CaCO 3 and extracted in 1 ml of 99% (v/v) acetone in darkness for 15 min under a nitrogen atmosphere at room temperature. The extract was centrifuged for 12 min at 4000 g. Extraction and centrifugation were repeated and the resulting supernatants combined and ltered through a 0.45 lm Nalgene lter. Xanthophyll-cycle pigments and chlorophylls were separated and quanti ed by HPLC, as described previously (Streb et al. 1997), except that a non-endcapped LiChrosphere-100-C18 reversed-phase column from Knauer was used. In HPLC separations of pigment extracts, a-tocopherol was detected and quanti ed with a uorescence detector (excitation at 295 nm; emission at 330 nm), as described by SchuÈ ep and Rettenmaier (1994). Contents of xanthophyll-cycle pigments and a-tocopherol were expressed on the basis of total chlorophyll content, as determined in the separation of the same sample. Light-dependent oxygen evolution of leaves was measured with a Hansatech (Kings Lynn, Norfolk, UK) leaf-disc electrode at a saturating photon ux in the presence of 5±10% CO 2, as described by Streb et al. (1997). Chlorophyll a uorescence was measured with a portable photosynthesis yield analyser (Mini-Pam; H. Walz, E eltrich, Germany). Initial uorescence (F o ) was determined after excitation of a dark-adapted leaf with weak modulated light in the ML-burst mode of the Mini-Pam to con rm that the measuring light caused no closure of PSII reaction centres. Maximum uorescence (F m ) was measured after a saturating light pulse of approximately 7000 lmol m )2 s )1 PAR. Actinic light of 500± 2000 lmol m )2 s )1 PAR was provided by an external halogen lamp in combination with a ventilated infrared lter (2060-M, 20 W; H. Walz). The leaf temperature was maintained between 22 C and 28 C. For measurements at low temperature, leaves were kept on an ice-cold support. For irradiation with 500 lmol m )2 s )1 PAR a cold light source (Schott KL 1500) with two glass- bre leads was used.the leaf temperature was 4 C. Saturating light pulses of 2 s duration were applied every 2 min during exposure to actinic light or darkness. Photoinhibition of PSII was determined as decrease of the ratio of variable to maximum chlorophyll a uorescence (F v /F m ) after a minimum of 10 min dark adaptation (Krause and Weis 1991). In some experiments F v / F m was measured after dark periods of up to 1 h. Approximations of the electron transport rate through PSII were calculated as (F m )F)/F m PAR by multiplying the quantum e ciency of PSII according to Genty et al. (1989) by the incident photon ux density of PAR and an average factor of 0.8 for leaf absorptance, and dividing by a factor of 2 to account for the sharing of absorbed photons between the two photosystems. In this equation, F is the actual and F m the corresponding maximum uorescence in light. The photochemical quenching (qp) and the non-photochemical quenching (qn) were calculated according to Schreiber et al. (1986) at the end of the actinic light irradiations and corrected for quenching of F o according to Bilger and Schreiber (1986). The approximate percentage of reduced primary electron acceptor of PSII (Q A ) was calculated as 1)qP according to Dietz et al. (1985). All experiments were repeated independently at least three times. Mean values with standard errors of the mean are presented. Results Functional properties of chloroplasts. Of the three alpine plant species, Homogyne alpina, Ranunculus glacialis and Soldanella alpina, that were used for our investigations, only R. glacialis allowed the isolation of chloroplasts with high yields. Therefore, only this plant was used for

4 316 P. Streb et al: Photoprotection in alpine plants Fig. 1. Comparison of the activities of PSII, PSI and whole-chain electron transport in isolated thylakoid membranes obtained from leaves of R. glacialis and R. acris. For PSII-mediated electron transport, phenyl-p-benzoquinone (PBQ) was used as electron acceptor; PSI was assayed with ascorbate (Asc) as electron donor and methyl viologen (MV ) as acceptor; for whole-chain electron transport (PSII + PSI), methyl viologen was used as acceptor a comparison of photochemical activities in vitro. The activities of PSI and PSII and whole-chain electron transport were assayed with thylakoids obtained from Percoll-gradient-puri ed intact chloroplasts from R. glacialis and compared with corresponding preparations from the lowland plant R. acris (Fig. 1). The in vitro activities of both photosystems were more than 50% higher in R. glacialis than in R. acris, and the capacity of whole-chain electron transport was almost twice as high. The ratio of PSII/PSI was not, however, signi cantly di erent in the two plant species, and did not therefore indicate any peculiar high-light acclimation in the alpine species. The ratio of measured electron transport rates of PSII/PSI amounted to 1.6 for R. glacialis and to 1.4 for R. acris. Previous investigations have indicated that the inactivation of the light-sensitive enzyme catalase in vitro is not only mediated by blue light absorbed by its heme prosthetic groups but may also occur as a result of photooxidative events initiated in chloroplasts when the enzyme is irradiated in a suspension together with the latter organelle (Feierabend and Engel 1986; Feierabend et al. 1996). Chloroplast-mediated inactivation of catalase is best demonstrated by irradiation of a mixture of intact chloroplasts and catalase with red light which is not absorbed by the catalase and did not inactivate the enzyme (Fig. 2). Rates of catalase inactivation were very similar in the presence of intact chloroplasts from the lowland plants Taraxacum o cinale or Secale cereale. According to additional investigations, chloroplast-mediated inactivation was only observed when appropriate electron acceptors for the photosynthetic electron transport were missing, especially when the plastoquinone compounds were not oxidized. Under such conditions reactive oxygen species, including superoxide, are generated and mediate the oxidative inactivation of catalase (data not shown). When intact isolated chloroplasts Fig. 2. Comparison of the inactivation of bovine liver catalase in the presence of intact isolated chloroplasts from leaves of S. cereale (e), T. o cinale (h), and R. glacialis (n) in red light. s, control with catalase in red light in the absence of chloroplasts; m, control with catalase and isolated chloroplasts from R. glacialis in darkness; n, control with catalase and isolated chloroplasts from T. o cinale in darkness Table 1. Comparison of the contents of a-tocopherol in the leaves of the alpine plants R. glacialis and S. alpina and in the primary leaves of S. cereale. Leaves of alpine plants were collected from eld sites, S. cereale was grown under controlled conditions for 6 d at 22 C (non-hardened) or for 5 weeks at 4 C (cold-hardened) in 96 lmol m )2 s )1 PAR continuous white light. The leaves of the alpine plants were exposed to sunlight for 3 h before extraction Plant species a-tocopherol [mmol (mol chlorophyll) )1 ] S. cereale, 22 C-grown S. cereale, 4 C-grown R. glacialis S. alpina from R. glacialis were irradiated together with catalase in red light, only very little inactivation of catalase was observed. This result suggests that R. glacialis chloroplasts had an improved ability to avoid the accumulation of reactive oxygen species that were detected through the inactivation of accompanying catalase. The content of a-tocopherol which represents the main scavenger for free radicals in thylakoid membranes was, however, extraordinarily low in leaves of R. glacialis (Table 1). While the content of a-tocopherol greatly increased when S. cereale leaves were cold-hardened by growing the plants at 4 C, the amount of a-tocopherol in R. glacialis leaves accounted for only about 1.5% of that found in S. alpina leaves and was even much lower than in non-hardened S. cereale leaves grown in low light at 22 C. Paraquat sensitivity. In order to assess and compare the capacities for antioxidative protection, paraquat tolerance was compared in leaves of the alpine plants H. alpina, R. glacialis and S. alpina and in those of the lowland plants R. acris and T. o cinale. As symptoms of oxidative damage, the bleaching of chlorophyll and the inactivation of catalase (see Streb et al. 1993) were measured at di erent concentrations of

5 P. Streb et al: Photoprotection in alpine plants 317 Fig. 3. Catalase activity and chlorophyll content (as percent of control without paraquat) in leaves of the alpine plants S. alpina (s), H. alpina (,), R. glacialis (h) and the lowland plants T. o cinale (r) and R. acris (m) after 24 h exposure to light of 1000 lmol m )2 s )1 PAR at 25 C in the presence of di erent paraquat concentrations paraquat (Fig. 3). In general, catalase was more sensitive to paraquat-induced photooxidation than chlorophyll. A striking di erence in paraquat-sensitivity was observed between the lowland plants R. acris and T. o cinale and the alpine species H. alpina and S. alpina. In the lowland plants, catalase activity was totally lost in the presence of 10 lm paraquat and in T. o cinale chlorophyll was also largely degraded at this concentration. In R. acris chlorophyll had a somewhat higher stability in the presence of paraquat. The alpine species H. alpina and S. alpina exhibited a much higher paraquat tolerance, with respect to photooxidation of both catalase and chlorophyll, than the lowland plants. At 100 lm paraquat they had still retained more than 50% of their chlorophyll contents. However, the alpine species R. glacialis was as sensitive to paraquat-induced photooxidation as the lowland plants. Chlorophyll degradation was even more paraquat-sensitive in R. glacialis than in R. acris (Fig. 3). In leaves of two alpine species which greatly di ered in their paraquat-tolerance, changes in the contents of the antioxidants ascorbate and glutathione were analyzed after incubation at di erent paraquat concentrations in the light (Fig. 4). Incubation of the leaves in light without paraquat did not greatly a ect the antioxidant contents, except for a slight increase in the content of total glutathione (GSH and GSSG, the reduced and oxidized forms, respectively) in R. glacialis. With increasing paraquat concentration, total ascorbate (reduced ascorbate + dehydroascorbate) as well as total glutathione (GSH + GSSG) were greatly degraded. However, this degradation occurred at a much lower Fig. 4. Comparison of the e ects of di erent paraquat concentrations on the contents of reduced ascorbate (AA ) and dehydroascorbate (DHA ), and of reduced (GSH ) and oxidized (GSSG ) glutathione in leaves of S. alpina and R. glacialis. Leaves were assayed before (untreated control) and after a 24-h exposure to light of 1000 lmol m )2 s )1 PAR at 25 C in the presence of paraquat. Di erent scales are used for the antioxidant contents of R. glacialis and S. alpina paraquat concentration in R. glacialis than in S. alpina. Thus the ascorbate content of S. alpina at 100 lm paraquat was still as high as in R. glacialis leaves in the absence of paraquat. In addition, both ascorbate and GSH were already almost totally oxidized at paraquat concentrations as low as 2 lm in R. glacialis, whereas in S. alpina leaves the percentage of reduced ascorbate was, even at 100 lm, higher than that of its oxidized form (Fig. 4). E ects of inhibitors interfering with zeaxanthin formation and photorespiration. The application of DTT has been introduced to inhibit the light-induced de-epoxidation of violaxanthin and was found to suppress non-photochemical uorescence quenching and thermal dissipation of excitation energy which often appeared to be correlated with the occurrence of zeaxanthin (Demmig- Adams et al. 1990; Demmig-Adams and Adams III 1996). After 14 h overnight incubation at 20 C in darkness, zeaxanthin was not detectable in leaves of R. glacialis but a considerable amount (13% of the total xanthophyll-cycle carotenoids) was still present in S. alpina leaves. Zeaxanthin and antheraxanthin together amounted to approximately 30% of the xanthophyllcycle pigments in S. alpina leaves after 14 h dark incubation. After exposure to light the contents of both zeaxanthin and antheraxanthin increased strongly in

6 318 P. Streb et al: Photoprotection in alpine plants Fig. 5. Comparison of the contents of zeaxanthin, antheraxanthin and violaxanthin in leaves of S. alpina and R. glacialis after treatment with DTT. After a 14-h preincubation in darkness (Dark), in the absence (Control) or presence of 30 mm DTT, leaves were exposed for 10 min to 1000 lmol m )2 s )1 PAR light at 25 C or for 3 h to sunlight either at ambient temperature or on ice R. glacialis as well as in S. alpina. Within 10 min exposure to strong light the de-epoxidation of violaxanthin had essentially reached its maximum (Fig. 5). However, also in light, both the amount of zeaxanthin per unit chlorophyll and its proportion among the total xanthophyll-cycle pigments remained much lower in R. glacialis than in S. alpina, as pointed out previously (Streb et al. 1997). In addition, the total contents of xanthophyll-cycle pigments declined during the light exposure in R. glacialis. Incubation of leaves in the presence of 30 mm DTT during the light exposures prevented the formation of zeaxanthin in R. glacialis and blocked its additional accumulation in S. alpina. A lower concentration of 3 mm DTT, which is usually applied in experiments, was not su cient to suppress zeaxanthin formation in these plants. The contents of zeaxanthin and antheraxanthin that were still present at the end of the dark period in S. alpina declined during the exposure to light in the presence of DTT, but zeaxanthin could not be totally depleted in these leaves. Particularly high levels of zeaxanthin and antheraxanthin were retained in the presence of DTT when the leaves were kept on ice during irradiation with sunlight (Fig. 5). At low light intensities the non-photochemical quenching of chlorophyll uorescence (qn) was considerably higher in S. alpina than in R. glacialis. In both plants it increased with the photon ux to a similar maximum level (Fig. 6). This dose-dependent Fig. 6. In uence of DTT on the reduction state of the primary electron acceptor of PSII Q A (1)qP), non-photochemical uorescence quenching (qn), and on the calculated electron transport rates through PSII in leaves of S. alpina (s, d) or R. glacialis (h, n) exposed to di erent light intensities in the absence (open symbols) or presence ( lled symbols) of 30 mm DTT. Measurements were performed at the end of a 10-min (500±1500 lmol m )2 s )1 PAR) or 30-min (2000 lmol m )2 s )1 ) irradiation with actinic light following a 14-h preincubation in darkness at 20 C in the absence or presence of 30 mm DTT increase of qn in light was diminished in both plant species in the presence of DTT; however, at 2000 lmol m )2 s )1 light in the presence of DTT, qn was nally higher in R. glacialis than in S. alpina. In S. alpina the proportion of reduced Q A, indicated as 1)qP, strongly increased and the calculated rate of electron transport through PSII declined in the presence of DTT at both low and high photon ux. In R. glacialis the primary acceptor Q A was under all conditions in a more oxidized state than in S. alpina. In untreated control leaves the proportion of reduced Q A was only about half as high as in S. alpina. Vice versa, the electron transport rate through PSII was always much higher in R. glacialis than in S. alpina and increased with the photon ux up to 2000 lmol m )2 s )1 PAR (Fig. 6). After treatment with DTT increases in the proportion of reduced Q A and inhibition of the electron transport through PSII were seen only at

7 P. Streb et al: Photoprotection in alpine plants 319 Fig. 8. Changes in the F v /F m ratios in leaves of R. glacialis and S. alpina during a 3-h exposure to sunlight at ambient temperature or on ice in the absence or presence of either 30 mm DTT or 1 mm PPT. The light exposures were preceded by a 14-h preincubation in darkness at 20 C on water or with 30 mm DTT, or a 2-h preincubation in weak room light at 20 C with 1 mm PPT. Control, measurements taken before exposure to sunlight. F v /F m ratios were determined after 10 min dark adaptation Fig. 7. Comparison of the e ects of DTT and PPT at low temperature on non-photochemical uorescence quenching (qn), the calculated electron transport rates through PSII, the reduction state of the primary electron acceptor Q A (1)qP), and the F v /F m ratios in leaves of S. alpina and R. glacialis. Leaves were preincubated at 20 C for 14 h in darkness with 30 mm DTT or for 2 h with 1 mm PPT in weak room light before the 30-min exposure to actinic light of 500 lmol m )2 s )1 PAR on ice (4±7 C). Controls were kept on water. For determinations of qn, electron transport and (1)qP), measurements were performed at the end of the 30-min irradiation period. For F v /F m the change induced by the 30-min irradiation is indicated. Measurements of F v /F m at the end of the light period were performed after an additional 10 or 30 min dark adaptation in order to document potential recovery in darkness higher light intensities exceeding 1000 lmol m )2 s )1 PAR in R. glacialis. When the leaves were kept at a low temperature on ice at a photon ux of 500 lmol m )2 s )1 PAR, which allowed di erences in Q A reduction to be recognized, both the reduction state of Q A and qn were higher than at 25 C in R. glacialis as well as in S. alpina, while the electron transport through PSII was strongly retarded. In the presence of DTT the electron transport rates and qn were reduced and the reduction state of Q A further increased at low temperature (Fig. 7). These changes were similar in both species, except that the electron transport rate through PSII was also at low temperature under all experimental conditions several-fold higher in R. glacialis than in S. alpina. During short irradiation periods, as applied in the experiments of Figs. 6 and 7, the F v /F m ratio was not yet markedly a ected by the presence of DTT at 500 lmol m )2 s )1 PAR and low temperature (Fig. 7). After 30 min at 2000 lmol m )2 s )1 and 25 C a more marked decline of 31% was induced by the application of DTT in S. alpina, while the decline in R. glacialis was only 14% (data not shown). After a 3-h exposure to sunlight the di erential e ects of DTT became more apparent (Fig. 8). In sunlight the decline of F v /F m was greatly enhanced in S. alpina when the leaves were incubated on ice or in the presence of DTT, while the latter treatments had only minor e ects in R. glacialis, when applied separately. Only when low temperature and DTT were applied simultaneously was there a more marked 28% decline of the F v /F m ratio also in R. glacialis. This was, however, still much smaller than the 60% decline observed under these conditions in S. alpina (Fig. 8). In order to suppress photorespiration, leaves were treated with PPT. After a 2-h preincubation with PPT at low-intensity room light, leaves of R. glacialis had lost 85%, and leaves of S. alpina 90% of their glutamine synthetase activity. In PPT-treated leaves photosynthetic oxygen evolution in the presence of saturating CO 2 was between 80±90% of the rate measured in untreated leaves, demonstrating that the inhibitor exerted no major direct e ects on photosynthesis under non-photorespiratory conditions (Table 2). During exposure to 1000 lmol m )2 s )1 PAR in the presence of PPT the proportions of reduced Q A and qn were increased. The relative extent of these changes was much larger in

8 320 P. Streb et al: Photoprotection in alpine plants Table 2. E ect of a 2-h treatment with 1 mm PPT on glutamine synthetase activity and on total photosynthetic oxygen evolution (at saturating CO 2 ) in leaves of R. glacialis and S. alpina. One mg chlorophyll is equivalent to a leaf area of )3 m 2 for R. glacialis and of )3 m 2 for S. alpina Plant species Glutamine Oxygen evolution synthetase [lmol h )1 (mg [nkat (g FW) )1 ] chlorophyll) )1 ] R. glacialis )PPT PPT S. alpina )PPT PPT ratio of about 50% within 3 h in both R. glacialis and S. alpina. Similar declines in the F v /F m ratio were also seen when treated leaves were, in addition, kept on ice. However, while in S. alpina the presence of PPT did not much further enhance the extent of photoinhibition that was also observed in its absence on ice, the decline in the F v /F m ratio seen in ice-incubated R. glacialis leaves was mainly induced by the presence of PPT (Fig. 8). The rate of light-saturated photosynthetic oxygen evolution in the presence of saturating CO 2 also declined during exposure to PPT in light. The decline in photosynthetic oxygen evolution, in general, slightly exceeded the decline in the F v /F m ratio (data not shown). R. glacialis than in S. alpina (Fig. 9), and at 25 C than at low temperature (Fig. 7). The most striking e ect of PPT was that it strongly reduced the electron transport rate through PSII in R. glacialis but not in S. alpina (Fig. 9). In R. glacialis the electron transport rate further declined during the light exposure in the presence of PPT but was only slightly a ected in S. alpina. Application of PPT induced a decline in the F v /F m ratio in R. glacialis as well as in S. alpina leaves when they were irradiated with 1000 lmol m )2 s )1 PAR at ambient temperature (Fig. 9). During the next approximately 30 min of a subsequent dark incubation a slow recovery from photoinhibition was observed in both plant species. Prolonged exposure to sunlight in the presence of PPT induced marked declines in the F v /F m Discussion Chilling in the light is harmful to green plants because the reoxidation of the photosynthetic electron transport chain, as indicated by the redox state of Q A (Havaux 1987; OÈ quist et al. 1993), is impaired, photooxidative reactions are enhanced (Wise 1995) and the repair of damaged proteins is suppressed. Photoinactivation of PSII and of catalase are particularly sensitive symptoms of photodamage that have been observed in many plants at chilling temperatures (OÈ quist et al. 1987; Feierabend et al. 1992; Huner et al. 1993). However, in the leaves of three alpine high-mountain plants that were previously investigated, PSII as well as catalase remained fairly stable at high irradiance and low temperature, even when translation was blocked by inhibitors, although Fig. 9A,B. Comparison of the e ects of PPT on the reduction state of the primary electron acceptor Q A (1)qP) of PSII, nonphotochemical uorescence quenching (qn), the calculated electron transport rates through PSII and the F v /F m ratios in leaves of R. glacialis (h, n) and S. alpina (s, d) during a 60-min exposure to actinic light of 1000 lmol m )2 s )1 PAR at 25 C. Leaves were preincubated for 2 h in weak room light at 20 C in the presence of 1 mm PPT or water (for controls) before the 60-min high light exposure. A Measurements of 1-qP and qn were performed at the end of the 60-min light exposure. B Electron transport rates were estimated and calculated every 10 min during the light exposure. Open symbols, controls without PPT; lled symbols, incubations with PPT. The F v /F m ratios were determined before and at the end of the 60-min light exposure and during a subsequent 1-h dark incubation, in order to document potential recovery in darkness

9 P. Streb et al: Photoprotection in alpine plants 321 both PSII and catalase activity could be readily photoinactivated after extraction from the leaves. An exception was that H. alpina contained a more stable catalase (Streb et al. 1997). E cient means for the avoidance of, or protection from, photoinhibitory damage may be provided by the thermal dissipation of excess excitation energy, scavenger systems for the removal of reactive oxygen species, and photorespiration which may serve as a sink for the consumption of excess reducing equivalents (Asada 1994; Foyer et al. 1994). The capacities and the functional relevance of di erent protective systems have now been compared in high-mountain plants, mainly in S. alpina and R. glacialis. Dissipation of excess absorbed light energy as heat by non-photochemical quenching of excited antenna chlorophyll depends on the generation of a transthylakoid proton gradient by photosynthetic electron transport and was found to be mostly correlated with the operation of the xanthophyll cycle and the light-dependent formation of zeaxanthin and antheraxanthin (Demmig-Adams and Adams 1996). The relevance of zeaxanthin formation for non-photochemical quenching and photoprotection is, however, still controversial, since the violaxanthin-to-zeaxanthin conversion did not correlate with the time course of heat emission (Havaux and Tardy 1997) and in Chlamydomonas mutants zeaxanthin formation was neither a necessary precondition for non-photochemical quenching nor required for survival in excessive light (Niyogi et al. 1997a). In addition, lutein also appeared to contribute to non-photochemical quenching and to the dissipation of excess absorbed light energy (Niyogi et al. 1997b). In S. alpina thermal energy dissipation by non-photochemical quenching mechanisms, potentially related to xanthophyll-cycle operation, appeared to be important for light stress tolerance, as reported for other coldacclimated plants (SchoÈ ner and Krause 1990; Adams et al. 1995; Haldimann et al. 1996; Leipner et al. 1997). Although the capacity for non-photochemical uorescence quenching of R. glacialis was also high, it was only to a minor extent related to the presence of zeaxanthin and antheraxanthin. In leaves of S. alpina the zeaxanthin and antheraxanthin contents were much higher than in those of R. glacialis, and a substantial amount of zeaxanthin (+antheraxanthin) was retained overnight even after extended dark periods at 20 C. Previously, the retention of high zeaxanthin levels in the dark had been observed after very cold nights in overwintering leaves and correlated with a decrease in the F v /F m ratio (Adams et al. 1995). Similarly, leaves of S. alpina that had survived over the winter always had a slightly reduced F v /F m ratio between 0.6 and 0.7 (Fig. 8), indicating that a weak chronic photoinhibition (Streb et al. 1997), and a high level of qn (Fig. 6) already accompanied the retention of zeaxanthin at moderate irradiance. Even though the pre-existing zeaxanthin could not be totally depleted, the inhibition of the additional light-induced violaxanthin-to-zeaxanthin conversion by DTT prevented the photon- ux-dependent increase of qn, greatly increased the proportion of reduced Q A, and induced marked photoinhibition of PSII in sunlight in S. alpina, thus emphasizing the signi cance of a xanthophyll-cycle-related energy dissipation for this plant. By contrast, in R. glacialis the e ects of DTT application on photosynthetic functions were much less marked. Whereas R. glacialis leaves remained totally depleted of zeaxanthin after dark incubation and in the presence of DTT, non-radiative excitation energy dissipation (qn) was, nevertheless, greatly increasing with the photon ux and appeared to be mostly independent of the operation of the xanthophyll cycle. That this zeaxanthin-independent excitation energy dissipation also contributed to photoprotection was suggested by the fact that the F v /F m ratio was only little a ected when R. glacialis leaves were treated with DTT at ambient temperature. Only when exposed to DTT under chilling conditions, did the proportion of reduced Q A increase more markedly (Fig. 7) and the F v / F m ratio decline in sunlight (Fig. 8) also in R. glacialis, although to a much lesser extent than in S. alpina. The striking di erences of paraquat tolerance between R. glacialis, which behaved like a non-acclimated plant, and the other alpine plants, S. alpina and H. alpina, provided compelling evidence that previously observed large di erences in the activities of antioxidative enzymes and of the contents of the antioxidants ascorbate and glutathione existing in these species (Streb et al. 1997) were indeed of substantial functional signi cance. It is unlikely that the di erences were related to major limitations of paraquat uptake since the application of PPT was e ective within 2 h in both plant species (Table 2) and substantial degradation of antioxidants indicated the presence of paraquat also in leaves of S. alpina. The low e ciency of the reactive oxygenscavenger systems of R. glacialis leaves was further accentuated by their low a-tocopherol content and by the strong oxidation and destruction of ascorbate and glutathione at low paraquat levels, although this is very exceptional among cold-acclimated plants. Usually high levels of antioxidative enzymes and antioxidants, as occurring in S. alpina, were observed in cold-acclimated leaves and regarded as essential for low-temperature acclimation (SchoÈ ner and Krause 1990; Wise 1995; Leipner et al. 1997). Photosynthetic performance of R. glacialis was most markedly a ected by PPT. Phosphinothricin inhibits glutamine synthetase and blocks the reassimilation of ammonia which appears to be a rate-limiting step for photorespiration (Kozaki and Takeba 1996). Photosynthesis is inhibited after PPT application, however, only under photorespiratory conditions. Detailed investigations have shown that the inhibition of photosynthesis was largely restored by the application of glutamine and did not result from toxic e ects of ammonia accumulation but was mainly related to the repression of photorespiration (Wendler and Wild 1990; Wendler et al. 1990). In transgenic tobacco plants in which the plastidic glutamine synthetase was either suppressed or overexpressed, the capacity for photorespiration changed with the level of expression of this enzyme. The resistance of these plants against photoinhibition of PSII and photooxidative bleaching increased in

10 322 P. Streb et al: Photoprotection in alpine plants proportion to the capacities of glutamine synthetase and photorespiration, thus emphasizing the protective role of the latter (Kozaki and Takeba 1996). Photorespiratory production of CO 2 may sustain the operation of the Calvin cycle under unfavorable conditions and serve as a means for the dissipation of excess excitation energy. Based on the above-mentioned results and conclusions, the very striking reduction of the apparent electron transport rates through PSII in R. glacialis, which was quite similar to that occurring in transgenic plants with reduced glutamine synthetase activity (Kozaki and Takeba 1996), indicates that photorespiration provided a major electron sink for R. glacialis, but appeared to be of minor relevance for S. alpina. Athough qn increased in the presence of PPT, this was obviously not su cient to prevent Q A becoming more strongly reduced and photoinhibition of PSII becoming enhanced also in R. glacialis. Surprisingly, PPT was also e ective at low temperature. While in S. alpina the low temperature induced some photoinhibition that was not much further enhanced by PPT, in R. glacialis a major decline in the F v /F m ratio was only observed when PPT was present under chilling conditions (Fig. 8). While photorespiration was mostly not expected to play a major role at low temperature (Wise 1995; Hurry et al. 1996; Streb et al. 1997), our present results suggest that in R. glacialis it may serve as a most essential photoprotective mechanism also at low temperature (Figs. 7, 8). Because of the low partial pressure of CO 2, photorespiration may be of particular importance for some high-mountain plants. It is very unlikely that a de ciency of amino acids and the resulting insu cient protein synthesis caused the photoinhibition induced by PPT, since PSII did not greatly depend on protein synthesis in these alpine plants (Streb et al. 1997; Shang and Feierabend 1998), and at low temperature, repair cannot have been of major importance within the short time periods. In conclusion, our comparative assessment of photoprotective mechanisms revealed greatly divergent strategies in two high-mountain plants. While S. alpina, like other cold-acclimated plants, was characterized not only by greatly enhanced capacities of scavenger systems for superoxide and H 2 O 2 but also by high levels of xanthophyll cycle pigments which already correlated with strong non-photochemical quenching at relatively low light intensities, this was not the case in R. glacialis. Leaves of R. glacialis sustained very high rates of electron transport, and this was in accord with high in-vitro activities of both photosystems, as compared to the low-elevation species R. acris, and with a high rate of photosynthetic oxygen evolution (Streb et al. 1997). However, in contrast to leaves of other plants acclimated to high irradiance (Anderson et al. 1995), the ratio of measured electron transport activities of PSII/PSI was not signi cantly increased and the chlorophyll a/b ratio (2.7) was relatively low (Streb et al. 1997). Since Q A was generally maintained in a highly oxidized state, strong electron sinks must be present in R. glacialis leaves also at low temperature. Consequently, non-radiative dissipation of excitation energy gained importance only at higher light intensities. The low capacities of the ascorbate-glutathione cycle and of the thylakoid-bound radical scavenger a-tocopherol, which were con rmed by the high paraquat sensitivity, exclude the possibility that the Mehler reaction might serve as major electron sink in R. glacialis under natural conditions, because the leaves would have su ered from severe photooxidative damage. Provided that no unknown alternative electron sink was available, the leaves of R. glacialis must maintain a highly e cient photosynthetic carbon metabolism that warrants the prompt consumption of reducing equivalents even under unfavorable conditions. Evidence has been presented that an increased capacity for carbon assimilation enables cold-hardened winter cereals to keep Q A oxidized and to avoid photoinhibition (Huner et al. 1993), whereas photorespiration appears to be insigni cant for these plants at low temperature (Hurry et al. 1996). Our present results suggest that photorespiration did provide a major electron sink for R. glacialis and was also essential for its photoprotection at low temperature; this possibility, however, needs to be further demonstrated by direct measurements. Additional adaptive mechanisms which are independent of carbon assimilation or photorespiration must, however, exist in the chloroplasts of R. glacialis. When isolated intact chloroplasts were irradiated without an electron acceptor in vitro the appearance of PSII photoinhibition was signi cantly delayed, relative to broken chloroplasts (Streb et al. 1997). Furthermore, the oxidative inactivation of catalase in red light, a process which is mediated by isolated chloroplasts from lowland plants and provides a means to detect the release of reactive oxygen by this organelle, was much lower in the presence of chloroplasts from R. glacialis. These chloroplasts must therefore be able to avoid the production of those reactive oxygens that were deleterious for catalase, or to detoxify them more e ciently than chloroplasts from lowland plants. The latter possibility is, however, less likely because the content of the radical scavenger a-tocopherol was extremely low in R. glacialis. Only the concentrations of ascorbate and glutathione were relatively high within the chloroplasts from R. glacialis, as compared to non-alpine plants (Streb et al. 1997), but not su cient to improve the general paraquat tolerance of the leaves. We thank the Deutsche Forschungsgemeinschaft, Bonn, for nancial support. Travel grants by the Dr. Senckenbergische Stiftung and the Herrmann Willkomm-Stiftung, Frankfurt am Main, are greatly appreciated. P.S., W.Sh. and J.F. are very grateful for the great hospitality and support which they received at the Station Alpine du Lautaret, Universite Joseph Fourier, in France. Phosphinothricin was kindly supplied by the AgrEvo GmbH, Frankfurt am Main. References Adams III WW, Hoehn A, Demmig-Adams B (1995) Chilling temperatures and the xanthophyll cycle. A comparison of warm-grown and overwintering spinach. Aust J Plant Physiol 22: 75±85